Role of airway smooth muscle cell phenotypes in airway tone and obstruction in guinea pig asthma model.

BACKGROUND: Airway obstruction (AO) in asthma is driven by airway smooth muscle (ASM) contraction. AO can be induced extrinsically by direct stimulation of ASM with contractile agonists as histamine, or by indirect provocation with antigens as ovalbumin, while the airway tone is dependent on intrinsic mechanisms. The association of the ASM phenotypes involved in different types of AO and airway tone in guinea pigs was evaluated. METHODS: Guinea pigs were sensitized to ovalbumin and challenged with antigen. In each challenge, the maximum OA response to ovalbumin was determined, and before the challenges, the tone of the airways. At third challenge, airway responsiveness (AR) to histamine was evaluated and ASM cells from trachea were disaggregated to determinate: (a) by flow cytometry, the percentage of cells that express transforming growth factor-ß1 (TGF-ß1), interleukin-13 (IL-13) and sarco-endoplasmic Ca2+ ATPase-2b (SERCA2b), (b) by RT-PCR, the SERCA2B gene expression, (c) by ELISA, reduced glutathione (GSH) and, (d) Ca2+ sarcoplasmic reticulum refilling rate by microfluorometry. Control guinea pig group received saline instead ovalbumin. RESULTS: Antigenic challenges in sensitized guinea pigs induced indirect AO, AR to histamine and increment in airway tone at third challenge. No relationship was observed between AO induced by antigen and AR to histamine with changes in airway tone. The extent of antigen-induced AO was associated with both, TGF-ß1 expression in ASM and AR degree. The magnitude of AR and antigen-induced AO showed an inverse correlation with GSH levels in ASM. The airway tone showed an inverse association with SERCA2b expression. CONCLUSIONS: Our data suggest that each type of AO and airway tone depends on different ASM phenotypes: direct and indirect AO seems to be sensitive to the level of oxidative stress; indirect obstruction induced by antigen appears to be influenced by the expression of TGF-ß1 and the SERCA2b expression level plays a role in the airway tone.

Regulation of Myosin Light-Chain Phosphatase Activity to Generate Airway Smooth Muscle Hypercontractility.

Smooth muscle is a central structure involved in the regulation of airway tone. In addition, it plays an important role in the development of some pathologies generated by alterations in contraction, such as hypercontractility and the airway hyperresponsiveness observed in asthma. The molecular processes associated with smooth muscle contraction are centered around myosin light chain (MLC) phosphorylation, which is controlled by a balance in the activity of myosin light-chain kinase (MLCK) and myosin light-chain phosphatase (MLCP). MLCK activation depends on increasing concentrations of intracellular Ca2+, while MLCP activation is independent of Ca2+. MLCP contains a phosphatase subunit (PP1c) that is regulated through myosin phosphatase target subunit 1 (MYPT1) and other subunits, such as glycogen-associated regulatory subunit and myosin-binding subunit 85 kDa. Interestingly, MLCP inhibition may contribute to exacerbation of smooth muscle contraction by increasing MLC phosphorylation to induce hypercontractility. Many pathways inhibiting MLCP activity in airway smooth muscle have been proposed and are focused on inhibition of PP1c, inhibitory phosphorylation of MYPT1 and dissociation of the PP1c-MYPT1 complex.

Airway Hyperresponsiveness in Asthma Model Occurs Independently of Secretion of ß1 Integrins in Airway Wall and Focal Adhesions Proteins Down Regulation.

The extracellular domains of some membrane proteins can be shed from the cell. A similar phenomenon occurs with ß1 integrins (α1ß1 and α2ß1) in guinea pig. The putative role of ß1 integrin subunit alterations due to shedding in airway smooth muscle (ASM) in an allergic asthma model was evaluated. Guinea pigs were sensitized and challenged with antigen. Antigenic challenges induced bronchoobstruction and hyperresponsiveness at the third antigenic challenge. Immunohistochemistry and immunoelectronmicroscopy studies showed that the cytosolic and extracellular domains of the ß1 integrin subunit shared the same distribution in airway structures in both groups. Various polypeptides with similar molecular weights were detected with both the cytosolic and extracellular ß1 integrin subunit antibodies in isolated airway myocytes and the connective tissue that surrounds the ASM bundle. Flow cytometry and Western blot studies showed that the expression of cytosolic and extracellular ß1 integrin subunit domains in ASM was similar between groups. An increment of ITGB1 mRNA in ASM was observed in the asthma model group. RACE-PCR of ITGB1 in ASM did not show splicing variants. The expression levels of integrin-linked kinase (ILK) and paxillin diminished in the asthma model, but not talin. The levels of phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr(696) increased in asthma model. Our work suggests that ß1 integrin is secreted in guinea pig airway wall. This secretion is not altered in asthma model; nevertheless, ß1 integrin cytodomain assembly proteins in focal cell adhesions in which ILK and paxillin are involved are altered in asthma model. J. Cell. Biochem. 117: 2385-2396, 2016. © 2016 Wiley Periodicals, Inc.

Antigen-induced airway hyperresponsiveness and obstruction is related to caveolin-1 expression in airway smooth muscle in a guinea pig asthma model.

BACKGROUND: Caveolin-1 is a fundamental signalling scaffold protein involved in contraction; however, the role of caveolin-1 in airway responsiveness remains unclear. We evaluated the relationship between caveolin-1 expression in airway smooth muscle (ASM) and antigen-induced airway responsiveness and obstruction in a guinea pig asthma model. METHODS: Airway obstruction in sensitised guinea pigs, induced by antigenic (ovalbumin) challenges administered every 10 days, was measured. Antigen-induced responsiveness to histamine and the expression of caveolin-1 and cavin 1, 2 and 3 were evaluated at the third ovalbumin challenge. The control group received saline solution instead of ovalbumin. RESULTS: After the first challenge, antigen exposure induced a transient airway obstruction and airway hyperresponsiveness, high levels of IL-4 and IL-5 in lung and airway globet cells proliferation at the third antigenic challenge. Caveolin-1 mRNA levels in total lung decreased in the experimental group compared with controls. Flow cytometric analysis of ASM from the experimental group showed a high number of cells expressing caveolin-1 compared with controls. This increase was confirmed by western blot. Airway obstruction and hyperresponsiveness correlated with the degree of increased caveolin-1 expression in ASM cells (P < 0.05; r = 0.69 and -0.52, respectively). The expression of cavins 1, 2 and 3 in ASM also increased in the experimental group compared to controls. Immunohistochemical findings reveal that differences in ASM caveolin-1 were not evident between groups. Nevertheless, a marked decrease in caveolin-1 and caspase 3 was observed in the pulmonary vascular smooth muscle of asthma model compared with controls. Histological analysis did not reveal differences in smooth muscles mass or subepithelial fibrosis levels in airways between groups. However, an enlargement of smooth muscle mass was observed in the pulmonary microvessels of experimental animals. This enlargement did not induce changes in pulmonary or systemic arterial pressures. CONCLUSIONS: Our data suggest that caveolin-1 expression in ASM has a crucial role in the development of antigen-induced airway obstruction and hyperresponsiveness in a guinea pig asthma model. In addition, the asthma model in guinea pigs appears to induce a contractile smooth muscle phenotype in the airways and a proliferative smooth muscle phenotype in pulmonary vessels.